Embryonic stem cells (ESCs) are pluripotent and have therapeutic potential in regenerative medicine. The pluripotent ESC identity is governed by a set of transcription factors centered on Oct4, Sox2 and Nanog together with a number of epigenetic regulators. To dissect the molecular basis for the pluripotency of ESCs, we have developed an in vivo biotinylation strategy for affinity purification of protein complexes (dubbed bioSAIP) and chromatin immunoprecipitation (dubbed bioChIP). These approaches have allowed us to delineate a protein interaction network surrounding Nanog, i.e., the Nanog interactome. The Nanog interactome is highly enriched for transcription factors including Oct4, which plays critical roles in stem cell pluripotency, somatic cell reprogramming and early development. It also encompasses several epigenetic regulatory pathways including Polycomb-repressive complex 1 (PRC1). Our preliminary data confirmed endogenous interaction between Oct4 and PRC1 proteins and uncovered unique PRC1 complexes in ESCs that comprise Rybp but not chromobox proteins (Cbxs). The overall objective of the proposed research is to use our well-established proteomics approaches coupled with mass spectrometry to establish the Oct4-centered "epigenetic interactome" encompassing the PRC1 interactome in mouse ESCs, and to elucidate molecular mechanism of PRC1 repression for stem cell maintenance. Our hypothesis is that the Oct4-centered "epigenetic interactome" links multiple critical epigenetic pathways to the Nanog interactome and comprises a unique PRC1 repression to maintain ESC identity. The proposed studies are: 1) to dissect the biochemical basis for epigenetic regulation of stem cell pluripotency;2) to establish the PRC1 interactome in ESCs;and 3) to decipher the mechanism of PRC1 function for target gene regulation and stem cell maintenance. These studies will not only lead to the discovery of novel pluripotency factors, but also illuminate the fundamental properties of stem cell pluripotency and the process of somatic cell reprogramming. Furthermore, it will provide a framework for exploring epigenetic mechanisms for stem cell pluripotency and finding ways to perturb epigenetic pathways for cell fate changes. PUBLIC HEALTH RELEVANCE: Embryonic stem cells (ESCs) can self renew indefinitely and differentiate into different cell types of a body, and are thus considered an unlimited, renewable source for cell-based therapies in regenerative medicine. We will dissect the biochemical basis for Oct4-centered epigenetic regulation of stem cell pluripotency, and decipher the mechanism of PRC1 function for target gene regulation and stem cell maintenance. This will provide a framework for understanding the epigenetic mechanisms of pluripotency (and cancer), and for exploring perturbation of epigenetic pathways to manipulate ESC fate changes for clinical application.